Ligation sequencing gDNA - Cas9 enrichment (SQK-CS9109)

Oxford Nanopore Technologies
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MinION: Protocol

Ligation sequencing gDNA - Cas9 enrichment (SQK-CS9109) V CAS_9106_v109_revH_16Sep2020

  • We advise all customers to read and consider the information in the "Targeted, amplification-free DNA sequencing using CRISPR/Cas" info sheet before starting this protocol
  • This protocol uses genomic DNA
  • Targeted cutting around specific genomic Regions of Interest (ROI) to achieve enrichment
  • Library preparation time is ~110 minutes
  • Fragment lengths are determined by the cut spacing, and not fragmentation
  • No PCR is required

For Research Use Only

This is a Legacy product This kit is soon to be discontinued. If customers require further support for any ongoing critical experiments using a Legacy product, please contact Customer Support via email: support@nanoporetech.com.

FOR RESEARCH USE ONLY

概要

  • We advise all customers to read and consider the information in the "Targeted, amplification-free DNA sequencing using CRISPR/Cas" info sheet before starting this protocol
  • This protocol uses genomic DNA
  • Targeted cutting around specific genomic Regions of Interest (ROI) to achieve enrichment
  • Library preparation time is ~110 minutes
  • Fragment lengths are determined by the cut spacing, and not fragmentation
  • No PCR is required

For Research Use Only

This is a Legacy product This kit is soon to be discontinued. If customers require further support for any ongoing critical experiments using a Legacy product, please contact Customer Support via email: support@nanoporetech.com.

Document version: CAS_9106_v109_revH_16Sep2020

1. Overview of the protocol

Features of using Cas9 targeted sequencing

We recommend CRISPR/Cas targeted sequencing if the user:

  • Wishes to sequence multiple human gene targets (up to 100 in a single panel) to high coverage (>100x) on a single MinION Mk1B flow cell
  • Wishes to sequence up to a 50 kb Region of Interest (ROI), using up to 100 target sites, in a single assay*
  • Has 1-10 µg of available gDNA
  • Wishes to gain insight into methylation patterns or other modified bases
  • Has gene targets that are highly repetitive, or wishes to evaluate the number of repeats in an expansion, where traditional amplification methods or sequencing-by-synthesis methods could yield a biased result
  • Wishes to sequence long gene targets in a single pass that are not amenable to long-range PCR (> 30 kb)
  • Optionally wishes to run multiple barcoded samples on a single flow cell.

* Note: This is known as ‘tiling’ an ROI.

Excision/single cut vs tiling approach

This protocol can be used for any probe design method (details of which can be found in the Targeted, amplification-free DNA sequencing using CRISPR/Cas info sheet). The recommended ‘excision approach’ and ‘single cut and read out’ method can follow the main flow of this protocol (shown in Figure 1). The ‘tiling’ approach requires the alterations to the protocol described in the Important (orange) boxes in the library preparation section. The main difference with the tiling approach is that both pools of probes need to be prepared separately (RNP complex formation, cleavage and dA-tailing and adapter ligation performed in separate tubes) then pooled during the final AMPure XP bead purification (shown in Figure 2).

Single pool Figure 1. Cas9 targeted sequencing protocol using the 'excision approach' or 'single cut and read out'.

tiling Figure 2. Cas9 targeted sequencing protocol using the 'tiling' approach

重要

Targeted, amplification-free DNA sequencing using CRISPR/Cas info sheet

For more details about the Cas9 targeted sequencing approach, how to design probes, and general expectations and guidance, please refer to the Targeted, amplification-free DNA sequencing using CRISPR/Cas info sheet. We strongly recommend that you read it before proceeding with your targeted sequencing experiments.

Introduction to the CRISPR/Cas protocol

This protocol describes how to carry out sequencing of genomic DNA using the Cas9 Sequencing Kit (SQK-CS9109) with enrichment of specific genomic regions using CRISPR/Cas.

For users with no previous nanopore sequencing experience, we recommend that a Lambda control experiment is completed first to become familiar with the technology.

Prepare for your experiment You will need to:

  • Extract your DNA, and check its length, quantity and purity. The quality checks performed during the protocol are essential in ensuring experimental success.
  • Ensure you have your sequencing kit, the correct equipment and third-party reagents
  • Download the software for acquiring and analysing your data
  • Check your flow cell to ensure it has enough pores for a good sequencing run

Library preparation Figure 1. shows and explains the biochemical steps used to prepare your barcoded DNA library using a Cas9 Sequencing Kit (SQK-CS9109), plus several third-party reagents.

Cas9 sequencing workflow

Figure 1. Cas-mediated PCR-free enrichment library preparation for sequencing.

  1. After DNA extraction, 5’ ends are dephosphorylated to reduce ligation of sequencing adapters to non-target strands.
  2. Cas9 ribonucleoprotein particles (RNPs), with bound crRNA and tracrRNA, are added to the genomic DNA, then bind and cleave the Region of Interest (ROI).
  3. dsDNA cleavage by Cas9 reveals blunt ends with ligatable 5’ phosphates.
  4. All of the DNA in the samples are dA-tailed, which prepares the blunt ends for barcode ligation.
  5. Sequencing adapters are ligated primarily to Cas9 cut sides, which are both 3’ dA-tailed and 5’ phosphorylated. The library preparation is cleaned up to remove excess adapters using AMPure XP beads and resuspended in Sequencing Buffer. Non-target molecules are not removed. The subsequent library preparation is added to the flow cell for sequencing.

Sequencing and analysis You will need to:

  • Start a sequencing run using the MinKNOW software, which will collect raw data from the device and convert it into basecalled reads
  • Start the EPI2ME software and select a workflow for further analysis (this step is optional)

Enrichment experiment steps and associated instructions

Step Instructions
1. Extract and prepare DNA Extraction methods
2. Design probes Targeted, amplification-free DNA sequencing using CRISPR/Cas - Probe design
3. QC input DNA Input DNA/RNA QC
4. Perform enrichment, and prepare sequencing library Cas Sequencing Kit protocol
5. Sequence on device Cas Sequencing Kit protocol
6. Take basecalled FASTQ files into analysis pipeline Targeted, amplification-free DNA sequencing using CRISPR/Cas - Evaluation of read-mapping characteristics from a Cas9 targeted sequencing experiment
7. Assess success of experiment and feed back into probe design and quality of input
重要

Compatibility of this protocol

This protocol should only be used in combination with:

  • Cas9 Sequencing Kit (SQK-CS9109)
  • R9.4.1 (FLO-MIN106) flow cells
  • Flow Cell Wash Kit (EXP-WSH004)

2. Equipment and consumables

材料
  • 5 µg high molecular weight genomic DNA (recommended); 1–10 µg (or 0.1–2 pmol) can be used accordingly.
  • Cas9 Sequencing Kit (SQK-CS9109)
  • Flow Cell Priming Kit (EXP-FLP002)
消耗品
  • S. pyogenes Cas9 Alt-R™ crRNAs (resuspended at 100 µM crRNA in TE pH 7.5)
  • S. pyogenes Cas9 tracrRNA (e.g., IDT Alt-R™, Cat # 1072532, 1072533 or 1072534) resuspended at 100 µM in TE pH 7.5
  • Alt-R® S. pyogenes HiFi Cas9 nuclease V3, 100 µg or 500 µg (IDT, Cat # 1081060 or # 1081061)
  • Nuclease-Free Duplex Buffer (IDT Cat # 11-01-03-01)
  • 1.5 ml Eppendorf DNA LoBind tubes
  • 0.2 ml 薄壁のPCRチューブ
  • Nuclease-free water (e.g. ThermoFisher, AM9937)
  • Agencourt AMPure XP beads (Beckman Coulter, A63881)
装置
  • 小型遠心機
  • Magnetic rack
  • ボルテックスミキサー
  • サーマルサイクラー
  • P1000 ピペット及びチップ
  • P200 ピペットとチップ
  • P100 ピペットとチップ
  • P20 ピペットとチップ
  • P10 ピペットとチップ
  • P2 ピペットとチップ
  • アイスバケツ（氷入り）
  • タイマー
オプション装置
  • Agilent Bioanalyzer (or equivalent)
  • Qubit fluorometer (or equivalent for QC check)
  • Eppendorf 5424 centrifuge (or equivalent)

For this protocol, you will need 1–10 µg or 0.1–2 pmol high molecular weight genomic DNA (5 µg recommended).

Cas9 Sequencing Kit contents (SQK-CS9109)

cs9109 v1

Name Acronym Cap colour No. of vials Fill volume per vial (μl)
Adapter Mix AMX Green 2 40
Ligation Buffer LNB Clear 2 200
Elution Buffer EB Black 1 200
Sequencing Buffer SQB Red 2 300
L Fragment Buffer LFB Orange 2 1,800
S Fragment Buffer SFB Grey 2 1,800
Loading Beads LB Pink 1 360
Phosphatase PHOS Brown tube, yellow label 1 50
TAQ Polymerase TAQ Brown tube, green label 1 15
SPRI Dilution Buffer SDB Brown tube, red label 1 1,200
T4 DNA Ligase LIG Brown tube, blue label 1 140
dATP dATP Brown tube, grey label 1 15
Reaction Buffer RB Brown tube, orange label 1 180

Flow Cell Priming Kit contents (EXP-FLP002)

FLP

Name Acronym Cap colour No. of vials Fill volume per vial (μl)
Flush Buffer FB Blue 6 1,170
Flush Tether FLT Purple 1 200
重要

Genomic DNA and its quality

Unsheared, high-molecular weight DNA, as isolated e.g., using the Qiagen Genomic DNA Kit, at ≥210 ng/µl by Qubit, and stored in TE (pH 8.0) or similar, or nuclease-free water.

Carryover of 1 mM EDTA from TE will not significantly affect this protocol; however, care should be taken to reduce other contaminants, such as detergents, phenol, chloroform, and salts.

ヒント

Wide-bore tips

Use wide-bore tips (or regular pipette tips with the narrow ends cut off) where possible to minimise shearing of long DNA.

Sourcing crRNA and tracrRNA

We recommend using synthetic crRNA and tracrRNA from IDT, which are of sufficient purity and carry modifications that confer stability and nuclease resistance. For this reason we caution against using single-guide RNAs (sgRNAs).

  • S. pyogenes Cas9 Alt-R™ crRNAs
  • S. pyogenes Cas9 tracrRNA (e.g. IDT Alt-R™, Cat # 1072532, 1072533 or 1072534)

Individual crRNAs and tracrRNA should be resuspended at 100 µM each in TE, pH 7.5, aliquoted to avoid freeze-thawing, and stored at –20° C for up to two weeks or –80° C if stored long-term. The crRNAs/tracrRNAs can be freeze-thawed a maximum of five times.

crRNAs may be pooled to make panels for generating multiple cuts in a single reaction. To pool crRNAs, we recommend dispensing equal volumes of each crRNA (up to 100 crRNAs, each at 100 µM) into a separate 1.5 ml Eppendorf DNA LoBind tube to make an equimolar crRNA mix.

We strongly recommend TE at pH 7.5, rather than pH 8.0, for the long-term stability of RNA oligos in storage.

3. Computer requirements and software

MinION Mk1B IT requirements

Sequencing on a MinION Mk1B requires a high-spec computer or laptop to keep up with the rate of data acquisition. For more information, refer to the MinION Mk1B IT requirements document.

MinION Mk1C IT requirements

The MinION Mk1C contains fully-integrated compute and screen, removing the need for any accessories to generate and analyse nanopore data. For more information refer to the MinION Mk1C IT requirements document.

MinION Mk1D IT requirements

Sequencing on a MinION Mk1D requires a high-spec computer or laptop to keep up with the rate of data acquisition. For more information, refer to the MinION Mk1D IT requirements document.

Software for nanopore sequencing

MinKNOW

The MinKNOW software controls the nanopore sequencing device, collects sequencing data and basecalls in real time. You will be using MinKNOW for every sequencing experiment to sequence, basecall and demultiplex if your samples were barcoded.

For instructions on how to run the MinKNOW software, please refer to the MinKNOW protocol.

EPI2ME (optional)

The EPI2ME cloud-based platform performs further analysis of basecalled data, for example alignment to the Lambda genome, barcoding, or taxonomic classification. You will use the EPI2ME platform only if you would like further analysis of your data post-basecalling.

For instructions on how to create an EPI2ME account and install the EPI2ME Desktop Agent, please refer to this link.

フローセルのチェックをしてください

シークエンシング実験を開始する前に、フローセルのポアの数を確認することを強くお勧めします。このフローセルの確認は、MinION/GridION/PromethIONの場合は代理店への到着から１２週間以内に行ってください。またはFlongle Flow Cellの場合は代理店への到着から4週間以内に行う必要があります。Oxford Nanopore Technologiesは、フローセルチェックの実施から2日以内に結果が報告され、推奨される保管方法に従っていた場合に、以下の表に記載されているナノポアの有効数に満たさない場合には、フローセルを交換します。 フローセルのチェックを行うには、Flow Cell Check documentの指示に従ってください。

Flow cell 保証する最小有効ポア数（以下の数未満のフローセルが交換対象となります）
Flongle Flow Cell 50
MinION/GridION Flow Cell 800
PromethION Flow Cell 5000

4. Preparing the Cas9 ribonucleoprotein complexes (RNPs)

消耗品
  • S. pyogenes Cas9 Alt-R™ crRNAs (resuspended at 100 µM crRNA in TE pH 7.5)
  • Alt-R® S. pyogenes HiFi Cas9 nuclease V3, 100 µg or 500 µg (IDT, Cat # 1081060 or # 1081061)
  • S. pyogenes Cas9 tracrRNA (e.g., IDT Alt-R™, Cat # 1072532, 1072533 or 1072534) resuspended at 100 µM in TE pH 7.5
  • Nuclease-Free Duplex Buffer (IDT Cat # 11-01-03-01)
  • 0.2 ml 薄壁のPCRチューブ
  • 1.5 ml Eppendorf DNA LoBind tubes
  • Nuclease-free water (e.g. ThermoFisher, AM9937)
装置
  • サーマルサイクラー
  • Ice bucket with wet ice
  • P200 ピペットとチップ
  • P10 ピペットとチップ
  • P2 ピペットとチップ
重要

Here, the Cas9 is loaded with crRNA and tracrRNA to form ribonucleoprotein complexes (RNPs) in preparation for the cleavage reaction.

重要

If using a 'tiling' approach

The formula below prepares a pool of RNPs for making multiple excisions in a single reaction. If using a tiling approach for probe design (a method for designing probes in two separate overlapping pools to cover a target region >20 kb), prepare 2x RNP complexes, one for each pool of crRNA probes. For more information about tiling, please refer to the Targeted, amplification-free DNA sequencing using CRISPR/Cas info sheet.

重要

RNP complex stability

Upon receipt, we recommend aliquoting individual probes or pools of crRNAs for storage, to minimise freeze-thawing.

Panels of RNPs may be formed ahead of time and stored at 4°C for up to a week, or at -80°C for up to a month with no discernible loss of activity. However, we recommend making a fresh RNP complex for every experiment if possible.

ヒント

crRNA and ribonucleoproteins (RNPs)

The following protocol applies to a single crRNA. For panels of multiple crRNAs, see “Notes on multiple crRNAs” below.

重要

Notes on multiple crRNAs

If you have validated a set of crRNA probes that are always run together, we recommend pooling all crRNA probes and then aliquoting that pool and freezing each aliquot at -80°C. Each time you run a Cas9 experiment and need to make an RNP complex, take out a crRNA pool aliquot and combine with the tracrRNA.

Pre-heat a thermal cycler to 95ºC.

Thaw an aliquot of Reaction Buffer (RB), mix by vortexing, and place on ice.

In an 1.5 ml Eppendorf DNA LoBind tube, pool the crRNA probes for each cleavage reaction by combining equal volumes of each crRNA probe, resuspended at 100 µM in TE (pH 7.5).

  • A single crRNA or many crRNA probes (up to ~100) may be used in a single cleavage reaction.
  • The crRNA probes may also be pre-mixed as an off-catalogue request from IDT.
  • For example, probes for the HTT gene, found here, can be used as an individual experiment or in addition to other probes as an in-run control.
  • Unused crRNA probe mix may be stored at -80ºC and minimal freeze thaw recommended.

Anneal the pooled crRNAs with tracrRNA in Nuclease-Free Duplex Buffer by assembling the following in a 0.2 ml thin-walled PCR tube, as follows:

Reagent Volume
Nuclease-Free Duplex Buffer (IDT) 8 µl
crRNA pool (100 µM, equimolar) 1 µl
tracrRNA (100 µM) 1 µl
Total 10 µl

Mix well by pipetting and spin down.

Using a thermal cycler heat the above reaction mix at 95ºC for 5 mins, then remove the tube from the thermal cycler and allow it to cool to room temperature, then spin down the tube to collect any liquid in the bottom of the tube.

  • Storage and reuse of the annealed mix is not recommended.

To form Cas9 RNPs, assemble the components in the table in an 1.5 ml Eppendorf DNA LoBind tube; this will form the annealed crRNA•tracrRNA, through pooling in the stated order:

Reagent Volume
Nuclease-free water 79.2 µl
Reaction Buffer (RB) 10 µl
Annealed crRNA•tracrRNA pool (10 µM) 10 µl (Step 4, above)
HiFi Cas9 (62 µM) 0.8 µl
Total 100 µl

Note: Refer to the tip below for scaling-down this ternary RNP mix.

Mix thoroughly by flicking the tube.

ヒント

The above step yields an excess amount of RNPs, but 10 µl are carried forwards for each reaction into the next target cleavage step. Any excess RNPs may be stored at 4ºC for up to a week. The reaction may be scaled, as shown in the below table.

Number of reactions 3 5 10
Components Volume (µl) Volume (µl) Volume (µl)
Annealed crRNA•tracrRNA pool (10 µM) (Step 1) 3 5 10
Reaction Buffer (RB) 3 5 10
Nuclease-free water 23.7 39.6 79.2
HiFi Cas9 (62 µM) 0.3 0.4 0.8
Total 30 50 100

Form the RNPs by incubating the tube at room temperature for 30 minutes, then return the RNPs on ice until required.

ヒント

Proceed to the next step (gDNA dephosphorylation) during the 30 min RNP incubation step.

5. Dephosphorylating genomic DNA

材料
  • 5 µg high molecular weight genomic DNA (recommended); 1–10 µg (or 0.1–2 pmol) can be used accordingly.
  • Phosphatase (PHOS)
消耗品
  • 1.5 ml Eppendorf DNA LoBind tubes
  • Nuclease-free water (e.g. ThermoFisher, AM9937)
  • 0.2 ml 薄壁のPCRチューブ
装置
  • サーマルサイクラー
  • P100 ピペットとチップ
  • P10 ピペットとチップ

This step reduces background reads by removing 5’ phosphates from non-target DNA ends.

重要

If using a 'tiling' approach

If using a tiling approach for probe design (a method for designing probes in two separate overlapping pools to cover a target region >20 kb), and have just produced 2x separate RNP complexes, users need to perform the dephosphorylation, Cas9 cleavage and adapter ligation step twice (one reaction per pool of probes). For more information about tiling, please refer to the Targeted, amplification-free DNA sequencing using CRISPR/Cas info sheet.

Prepare the DNA in nuclease-free water

  • Transfer 1-10 μg (with 5 μg recommended) genomic DNA into a 0.2 ml thin-walled PCR tubes
  • Adjust to 24 µl with nuclease-free water
  • Mix thoroughly by flicking the tube avoiding unwanted shearing
  • Spin down briefly in a microfuge

Mix the Phosphatase (PHOS) in the tube by pipetting up and down. Ensure that it is at room temperature before use.

Assemble the following components in a clean 0.2 ml thin-walled PCR tube:

Reagent Volume
Reaction Buffer (RB) 3 µl
HMW genomic DNA (at ≥ 210 ng/µl)* 24 µl
Total 27 µl
  • Note: For an initial test, we recommend 5 µg genomic DNA input. Preparing input DNA step yields ~100-2000x target coverage. Target coverage scales linearly with input amount, so the input amount may be reduced accordingly if lower throughput is acceptable.

Ensure the components are thoroughly mixed by pipetting, and spin down.

Add 3 µl of PHOS to the tube.

Mix gently by flicking the tube, and spin down.

Using a thermal cycler, incubate at 37ºC for 10 minutes, 80ºC for 2 minutes then hold at 20ºC (room temperature).

6. Cleaving and dA-tailing target DNA

材料
  • 5 µg high molecular weight dephosphorylated genomic DNA (recommended); 1 - 10 µg (or 0.1-2 pmol) can be used accordingly.
  • crRNA-tracrRNA-Cas9 ribonucleoprotein complexes (RNPs)
  • Taq Polymerase (TAQ)
  • dATP (dATP)
消耗品
  • 1.5 ml Eppendorf DNA LoBind tubes
  • 0.2 ml 薄壁のPCRチューブ
装置
  • サーマルサイクラー
  • Ice bucket with wet ice
  • ボルテックスミキサー
  • P100 ピペットとチップ
  • P20 ピペットとチップ
  • P10 ピペットとチップ
  • P2 ピペットとチップ

In this step, Cas9 RNPs (see 'Preparing the Cas9 ribonucleoprotein complexes') and Taq polymerase are added to the dephosphorylated genomic DNA sample.

This process cleaves target and dA-tails all available DNA ends in one step, activating the Cas9 cut sites for ligation.

Thaw the dATP tube, vortex to mix thoroughly and place on ice.

Spin down and place the tube of Taq Polymerase (TAQ) on ice.

To the PCR tube containing 30 µl dephosphorylated DNA sample, add:

Reagent Volume
Dephosphorylated genomic DNA sample 30 µl
Cas9 RNPs 10 µl
dATP 1 µl
Taq Polymerase (TAQ) 1 µl
Total 42 µl

Carefully mix the contents of the tube by gentle inversion, then spin down and place the tube in the thermal cycler.

Using the thermal cycler, incubate at 37ºC for 15-60 minutes*, then 72ºC for 5 minutes and hold at 4ºC or return to tube to ice.

重要

*The Cas9 enzyme is active at 37ºC, and denatured at 72ºC. We recommend a 15 minute cut time by default. Longer 37ºC incubations may increase the amount of off-target reads without increasing the yield of on-target reads, while shorter incubations may result in incomplete target cleavage. However, some regions may benefit from a longer incubation at 37ºC.

7. Adapter ligation

材料
  • Ligation Buffer (LNB)
  • Adapter Mix (AMX)
  • T4 Ligase (LIG)
消耗品
  • 1.5 ml Eppendorf DNA LoBind tubes
  • Agencourt AMPure XP beads (Beckman Coulter™, A63881)
装置
  • Ice bucket with wet ice
  • ボルテックスミキサー
  • P1000 ピペット及びチップ
  • P100 pipette
  • P20 ピペットとチップ

Here, AMX adapters from the Cas Sequencing Kit (SQK-CS9109) are ligated to the ends generated by Cas9 cleavage.

Thaw the Ligation Buffer (LNB) at room temperature, spin down and mix by pipetting. Due to viscosity, vortexing this buffer is ineffective. Place on ice immediately after thawing and mixing.

Carefully transfer the contents of the 0.2 ml thin-walled PCR tube to a fresh 1.5 ml Eppendorf DNA LoBind Tube using a wide-bore pipette tip.

Thaw an aliquot of Adapter Mix (AMX), mix by flicking the tube, pulse-spin to collect the liquid in the bottom of the vial, then return the vial to ice.

Bring the AMPure XP beads to room temperature.

Assemble the following at room temperature in a separate 1.5 ml Eppendorf DNA LoBind Tube, adding Adapter Mix (AMX) last, before you are ready to begin the ligation:

Reagent Volume
Ligation Buffer (LNB) 20 µl
Nuclease-free water 3 µl
T4 Ligase (LIG) 10 µl
Adapter Mix (AMX) 5 µl
Total 38 µl

Mix by pipetting the above ligation mix thoroughly. Ligation Buffer (LNB) is very viscous, so the adapter ligation mix needs to be well-mixed.

IMPORTANT: Add 20 µl of the adapter ligation mix to the cleaved and dA-tailed sample. Mix gently by flicking the tube. Do not centrifuge the sample at this stage. Immediately after mixing, add the remainder of the adapter ligation mix to the cleaved and dA-tailed sample, to yield an 80 µl ligation mix.

Ensure the components are thoroughly mixed by pipetting, and spin down.

Incubate the reaction for 10 minutes at room temperature.

重要

DNA precipitation

A white precipitate may form upon addition of the adapter ligation mix to the dA-tailed DNA. Adding the ligation mixture in two parts helps to reduce precipitation. However, the presence of a precipitate does not indicate failure of ligation of the sequencing adapter to target molecule ends.

8. AMPure XP bead purification

材料
  • Long Fragment Buffer (LFB)
  • Short Fragment Buffer (SFB)
  • Elution Buffer (EB)
  • SPRI Dilution Buffer (SDB)
消耗品
  • Agencourt AMPure XP beads (Beckman Coulter™, A63881)
  • 1.5 ml Eppendorf DNA LoBind tubes
装置
  • P1000 ピペット及びチップ
  • P200 ピペットとチップ
  • P20 ピペットとチップ
  • 1.5 mlエッペンドルフチューブに最適のマグネット式ラック
  • Eppendorf 5424 centrifuge (or equivalent)

This step removes excess unligated adapters and other short DNA fragments, and concentrates and buffer-exchanges the library in preparation for sequencing.

重要

If using a 'tiling' approach

Complete steps 1 and either 2 or 3 depending on the DNA fragment lengths you wish to retain. Then pool the samples together into a single tube, before performing steps 4, 5, and 6 with the modified volumes for your pooled sample. This includes 1 volume (160 µl) of SPRI Dilution Buffer (SDB) and 0.3X (96 µl) of AMPure XP beads. For more information about tiling, please refer to the Targeted, amplification-free DNA sequencing using CRISPR/Cas info sheet.

Thaw the Elution Buffer (EB) and SPRI Dilution Buffer (SDB) at room temperature, mix by vortexing, spin down and place on ice.

To enrich for DNA fragments of 3 kb or longer, thaw one tube of Long Fragment Buffer (LFB) at room temperature, mix by vortexing, spin down and place on ice.

To retain DNA fragments of all sizes, thaw one tube of Short Fragment Buffer (SFB) at room temperature, mix by vortexing, spin down and place on ice.

Add 1 volume (80 µl) of the SPRI Dilution Buffer (SDB) to the ligation mix. Mix gently by flicking the tube.

Resuspend the AMPure XP beads by vortexing.

Add 0.3x volume (48 µl) of AMPure XP Beads to the ligation sample. The volume of beads is calculated based on the volume after the addition of SDB. Mix gently by inversion. If any sample ends up in the lid, spin down the tube very gently, keeping the beads suspended in liquid.

Incubate the sample for 10 minutes at room temperature. Do not agitate or pipette the sample.

サンプルをスピンダウンし、マグネット上でペレット化します。チューブをマグネットの上に置き、無色透明になったら上清をピペットで取り除きます。

Wash the beads by adding either 250 μl Long Fragment Buffer (LFB) or 250 μl Short Fragment Buffer (SFB), depending on the size of your target molecule. Flick the beads to resuspend, then return the tube to the magnetic rack and allow the beads to pellet. Remove the supernatant using a pipette and discard.

Repeat the previous step.

Spin down and place the tube back on the magnet. Pipette off any residual supernatant. Allow to dry for ~30 seconds, but do not dry the pellet to the point of cracking.

Remove the tube from the magnetic rack and resuspend pellet in 13 µl Elution Buffer (EB). Incubate for 10 minutes at room temperature.

Note: For targets >30 kb, we recommend increasing the elution time to 30 minutes.

Pellet the beads on a magnet until the eluate is clear and colourless.

Remove and retain 12 µl of eluate which contains the DNA library in a clean 1.5 ml Eppendorf DNA LoBind tube.

  • Dispose of the pelleted beads
最終ステップ

The prepared library is used for loading onto the flow cell. Store the library on ice until ready to load.

オプショナルステップ

If quantities allow, the library may be diluted in Elution Buffer (EB) for splitting across multiple flow cells.

Additional buffer for doing this can be found in the Sequencing Auxiliary Vials expansion (EXP-AUX001), available to purchase separately. This expansion also contains additional vials of Sequencing Buffer (SQB) and Loading Beads (LB), required for loading the libraries onto flow cells.

ヒント

Library storage recommendations

We recommend storing libraries in Eppendorf DNA LoBind tubes at 4°C for short term storage or repeated use, for example, reloading flow cells between washes. For single use and long-term storage of more than 3 months, we recommend storing libraries at -80°C in Eppendorf DNA LoBind tubes. For further information, please refer to the DNA library stability Know-How document.

9. Priming and loading the SpotON flow cell

材料
  • Flow Cell Priming Kit (EXP-FLP002)
  • Loading Beads (LB)
  • Sequencing Buffer (SQB)
消耗品
  • 1.5 ml Eppendorf DNA LoBind tubes
  • Nuclease-free water (e.g. ThermoFisher, AM9937)
装置
  • MinION device
  • MinIONとGridIONのFlow Cell ライトシールド
  • P1000 ピペット及びチップ
  • P100 ピペットとチップ
  • P20 ピペットとチップ
  • P10 ピペットとチップ
  • SpotON Flow Cell

Thaw the Sequencing Buffer (SQB), Loading Beads (LB), Flush Tether (FLT) and one tube of Flush Buffer (FB) at room temperature before mixing the reagents by vortexing, and spin down at room temperature.

To prepare the flow cell priming mix, add 30 µl of thawed and mixed Flush Tether (FLT) directly to the tube of thawed and mixed Flush Buffer (FB), and mix by vortexing at room temperature.

Open the MinION Mk1B lid and slide the flow cell under the clip.

Press down firmly on the flow cell to ensure correct thermal and electrical contact.

Flow Cell Loading Diagrams Step 1a

Flow Cell Loading Diagrams Step 1b

オプショナルステップ

ライブラリーをロードする前にフローセルチェックを行い、使用可能なポアの数を把握して下さい。

フローセルが以前にチェックされている場合は、このステップを省略できます。

詳細については、MinKNOWプロトコルのフローセルチェックの手順 flow cell check instructionsを参照してください。

Slide the priming port cover clockwise to open the priming port.

Flow Cell Loading Diagrams Step 2

重要

フローセルからバッファーを引き上げる際には注意してください。20～30μl以上は除去せず、ポアのアレイ全体が常にバッファーで覆われていることを確認して下さい。アレイに気泡が入ると、ポアに不可逆的なダメージを与える可能性があります。

プライミングポートを開けた後に、カバーの下に小さな気泡がないかを確認して下さい。気泡を取り除くために少量の液を引き上げます。

  1. P1000ピペットを200 µ Lに設定して下さい。
  2. ピペットの先端をプライミングポートに差し込みます。
  3. 目盛りが220-230 ulと表示されるまでダイヤルを回して、20-30 ulを吸い上げるか、少量のバッファーがピペットの先端に入るのが見えるまでダイヤルを回します。

(注： プライミングポートからセンサーアレイ全体にバッファーがあることを確認してください。

Flow Cell Loading Diagrams Step 03 V5_JP

気泡が混入しないように、プライミングポートからフローセルにプライミングミックスを800µl注入し、 5分間待ちます。この５分間の間に、以下の手順でライブラリーをロードする準備をして下さい。

Flow Cell Loading Diagrams Step 04 V5_JP

Thoroughly mix the contents of the Loading Beads (LB) by pipetting.

重要

The Loading Beads (LB) tube contains a suspension of beads. These beads settle very quickly. It is vital that they are mixed immediately before use.

In a new tube, prepare the library for loading as follows:

Reagent Volume per flow cell
Sequencing Buffer (SQB) 37.5 µl
Loading Beads (LB), mixed immediately before use 25.5 µl
DNA library 12 µl
Total 75 µl

Note: Load the library onto the flow cell immediately after adding the Sequencing Buffer II (SBII) and Loading Beads II (LBII) because the fuel in the buffer will start to be consumed by the adapter.

フローセルのプライミングを完了させます。

  1. SpotON サンプルポートカバーをゆっくりと持ち上げ、SpotON サンプルポートにアクセスできるようにします。
  2. 200μlのプライミングミックスをフローセルのプライミングポート（SpotONサンプルポートではありません）に気泡が入らないように注入します。

Flow Cell Loading Diagrams Step 5_JP

Flow Cell Loading Diagrams Step 06 V5_JP

調製したライブラリーは、ロードする直前にピペッティング混合して下さい。

Add 75 μl of sample to the flow cell via the SpotON sample port in a dropwise fashion. Ensure each drop flows into the port before adding the next.

Flow Cell Loading Diagrams Step 07 V5

SpotONサンプルポートカバーをゆっくりと元に戻し、バング（カバーの先）がSpotONポートに入ることを確認し、プライミングポートを閉じます。

Step 8 update_JP

Flow Cell Loading Diagrams Step 9_JP

重要

最適なシークエンス出力を得るために、ライブラリーがロードされたすぐにライトシールドをフローセルに取り付けてください。

ライブラリーがフローセル上にある状態では（ウォッシングやリロードのステップを含める）、フローセルにライトシールドを付けたままにしておくことを推奨します。ライトシールドは、ライブラリーがフローセルから除去された時点で取り外すことができます。

ライトシールドを以下のようにフローセルに設置して下さい。

  1. ライトシールドの先端を慎重にクリップに当てます。 (注： ライトシールドをクリップの下に無理に押し込まないでください。

  2. ライトシールドをフローセルにゆっくりと下ろします。ライトシールドは、フローセルの上部全体を覆うようにSpotONカバーの周囲に取り付けます。

J2264 - Light shield animation Flow Cell FAW optimised-Japanese step10

注意

MinIONフローセルライトシールドは、フローセルに固定されていないため、取り付け後の取り扱いには注意が必要です。

最終ステップ

デバイスの蓋を閉め、MinKNOWでシークエンスランをセットします。

10. Data acquisition and basecalling

Overview of nanopore data analysis

For a full overview of nanopore data analysis, which includes options for basecalling and post-basecalling analysis, please refer to the Data Analysis document.

How to start sequencing

The sequencing device control, data acquisition and real-time basecalling are carried out by the MinKNOW software. Please ensure MinKNOW is installed on your computer or device. There are multiple options for how to carry out sequencing:

1. Data acquisition and basecalling in real-time using MinKNOW on a computer

Follow the instructions in the MinKNOW protocol beginning from the "Starting a sequencing run" section until the end of the "Completing a MinKNOW run" section.

2. Data acquisition and basecalling in real-time using the MinION Mk1B/Mk1D device

Follow the instructions in the MinION Mk1B user manual or the MinION Mk1D user manual.

3. Data acquisition and basecalling in real-time using the MinION Mk1C device

Follow the instructions in the MinION Mk1C user manual.

4. Data acquisition and basecalling in real-time using the GridION device

Follow the instructions in the GridION user manual.

5. Data acquisition and basecalling in real-time using the PromethION device

Follow the instructions in the PromethION user manual or the PromethION 2 Solo user manual.

6. Data acquisition using MinKNOW on a computer and basecalling at a later time using MinKNOW

Follow the instructions in the MinKNOW protocol beginning from the "Starting a sequencing run" section until the end of the "Completing a MinKNOW run" section. When setting your experiment parameters, set the Basecalling tab to OFF. After the sequencing experiment has completed, follow the instructions in the Post-run analysis section of the MinKNOW protocol.

重要

When selecting the sequencing kit in MinKNOW, please choose SQK-CAS109 instead of SQK-LSK109.

Understanding Cas enrichment

The Duty Time feature in the MinKNOW software can be used to judge the quality of your experiment. The duty time plot shows the distribution of channel states over time, grouped by time chunks, or 'buckets'. The basic view shows the five main channel states: Sequencing, Pore, Recovering, Inactive, and Unclassified. Clicking the "More" button shows a more detailed breakdown of channel states.

It is recommended to observe the duty time plot populating over the first 30 min-1 hr of the sequencing run. By this time, the channel state distribution will give an indication whether the DNA library is of a good quality, and whether the flow cell is performing well.

Note: The Duty Time plots will be noticeably different to a conventional SQK-LSK109 run. A much smaller percentage of pores will be observed as Sequencing/Strand.

If Active Channel Selection is enabled during the run, the software instantly switches to a new channel in the group if a channel is in the “Saturated” or “Multiple” state, or after ~5 minutes if a channel is “Recovering”. This feature maximises the number of channels sequencing at the start of the experiment, however this may also result in an artificially high number of "Sequencing" or "Pore" channels in the duty time plot. For this reason, we recommend referring to the Mux Scan Results plot, which shows the true distribution of channel states at the point of the most recent mux scan.

Understanding Duty Time plots during a Cas9 targeted sequencing run

As discussed above, the user should expect a lower proportion of pores in Sequencing compared to a standard SQK-LSK109 run, while the total number of available pores should be roughly consistent between a Cas9 targeted sequencing experiment and SQK-LSK109 experiment.

Throughput duty time

FLO-MIN106 Duty time plot for a Cas9 targeted sequencing experiment using a human gene. From the Duty Time plot, there is an equivalent number of active pores between a SQK-LSK109 run and Cas9 taregeted sequencing run. In a Cas9 experiment, the sequencing pore is roughly 5-15% (light green) of the total number of pores.

11. Downstream analysis

Post-basecalling analysis

There are several options for further analysing your basecalled data:

1. Bioinformatics tutorials

For more in-depth data analysis, Oxford Nanopore Technologies offers a range of bioinformatics tutorials, which are available in the Bioinformatics resource section of the Community. The tutorials take the user through installing and running pre-built analysis pipelines, which generate a report with the results. The tutorials are aimed at biologists who would like to analyse data without the help of a dedicated bioinformatician, and who are comfortable using the command line.

2. Research analysis tools

Oxford Nanopore Technologies' Research division has created a number of analysis tools, which are available in the Oxford Nanopore GitHub repository. The tools are aimed at advanced users, and contain instructions for how to install and run the software. They are provided as-is, with minimal support.

3. Community-developed analysis tools

If a data analysis method for your research question is not provided in any of the resources above, please refer to the Community-developed data analysis tool library. Numerous members of the Nanopore Community have developed their own tools and pipelines for analysing nanopore sequencing data, most of which are available on GitHub. Please be aware that these tools are not supported by Oxford Nanopore Technologies, and are not guaranteed to be compatible with the latest chemistry/software configuration.

12. フローセルの再利用と返却

材料
  • Flow Cell Wash Kit (EXP-WSH004)

シークエンス実験終了後、フローセルを再利用する場合は、Flow Cell Wash Kitのプロトコールに従い、洗浄したフローセルを2～8℃で保管してください。

Flow Cell Wash Kit protocolは、Nanoporeコミュニティーで入手できます。

ヒント

運転を停止したらできるだけ早くフローセルをウォッシュすることをお勧めします。しかし、これが不可能な場合はフローセルをデバイスに入れたまま、翌日にウォッシュをして下さい。

または、返送手順に従って、オックスフォード・ナノポアに返送してください。

フローセルの返却方法は hereをご覧ください。

(注： 製品を返却する前に、すべてのフローセルを脱イオン水で洗浄する必要があります。

重要

シークエンシング実験に関して問題が発生した場合や質問がある場合には、このプロトコルのオンライン版にあるトラブルシューティングガイドを参照してください。

13. DNA/RNA抽出、およびライブラリ調製時の問題点

以下は、最もよく起こる問題のリストであり、いくつかの原因と解決策が提案されています。

Nanopore Community Support セクションにFAQをご用意しています。

ご提案された解決策を試しても問題が解決しない場合は、テクニカルサポートに電子メール (support@nanoporetech.com)または LiveChat in the Nanopore Communityでご連絡ください。

サンプルの品質が低い

問題点 この問題が生じた可能性のある原因 解決策とコメント
DNAの純度が低い（DNAのOD 260/280のナノドロップ測定値が1.8未満およびOD 260/230が2.0～2.2未満） DNA抽出で必要な純度が得られていない 夾雑物の影響は、 Contaminants に示されています。コンタミネーションをもたらさないために別の抽出方法extraction method をお試しください。.

追加のSPRIクリーンアップステップの実施を検討して下さい。
低いRNA インテグリティー（RNA Integrity Number: <9.5 RIN、またはrRNAバンドがゲル上でスメアになっている） 抽出中にRNAが分解された 別のRNA抽出方法 RNA extraction methodを試してください。RINの詳細については、 RNA Integrity Number の資料を参照してください。詳細については、 DNA/RNA Handling のページをご覧ください。
RNAのフラグメントが予想より短い 抽出中にRNAが分解された 別のRNA抽出方法 RNA extraction methodを試してください。 RINの詳細については、 RNA Integrity Number の資料を参照してください。詳細については、DNA/RNA Handling のページをご覧ください。

RNAを扱う際には、RNaseフリーの環境で作業し、実験器具もRNaseフリーにしておくことをお勧めします。

AMPureビーズクリーンアップ後のDNA回収率が低い

問題点 この問題が生じた可能性のある原因 解決策とコメント
低回収率 AMPureビーズとサンプルの比率が予想していたのよりも低いことによるDNAの損失 1. AMPureビーズはすぐに沈降するため、サンプルに添加する前によく再懸濁させてください。

2. AMPureビーズ対サンプル比が0.4:1未満の場合、どのようなサイズのDNA断片でもクリーンアップ中に失われます。
低回収率 DNA断片が予想よりも短い サンプルに対するAMPureビーズの比率が低いほど、短い断片に対する選択が厳しくなります。 アガロースゲル(または他のゲル電気泳動法)上でインプットDNAの長さを設定してから、使用するAMPureビーズの適切な量を計算してください。 SPRI cleanup
エンドプレップ後の収率が低い 洗浄ステップで使用したエタノール濃度が低い（70%未満）。 エタノールが70%未満の場合、DNAは洗浄中にビーズから溶出されます。必ず正しい濃度（％）のエタノールを使用してください。

14. Issues during the sequencing run

以下は、最もよく起こる問題のリストであり、いくつかの原因と解決策が提案されています。

Nanopore Community Support セクションにFAQをご用意しています。

ご提案された解決策を試しても問題が解決しない場合は、テクニカルサポートに電子メール (support@nanoporetech.com)または LiveChat in the Nanopore Communityでご連絡ください。

シークエンス開始時のポアがフローセルチェック後よりも少ない場合

問題点 予想される原因 解決策とコメント
MinKNOWのフローセルチェックで確認されたポアの数より、シークエンシング開始時のポア数が少なく表示された。 ナノポアアレイに気泡が入ってしまった。 フローセルチェックをした後、フローセルをプライミングする前に、プライミングポート付近の気泡を取り除くことが必要です。 気泡を取り除かないと、気泡がナノポアアレイに移動し、空気に触れたたナノポアが不可逆的なダメージを負った可能性がある。これを防ぐための最適な方法が、 this videoで紹介されています。
MinKNOWのフローセルチェックで確認されたポアの数より、シークエンシング開始時のポア数が少なく表示された。 フローセルがデバイスに正しく挿入されていない。 シークエンスランを停止し、フローセルをシークエンス装置から取り出します。次に再度フローセルを挿入し、装置にしっかりと固定され、目標温度に達していることを確認します。GridIONやPromethIONの場合は別のフローセルの位置をお試しください。
MinKNOWのフローセルチェックで確認されたポアの数より、シークエンシング開始時のポア数が少なく表示された。 ライブラリー内の汚染物質がポアを失活させたり塞いだりしている。 フローセルチェックの際のポア数は、フローセル保存バッファー中のQC用のDNA分子を用いて計測されます。シークエンシングの開始時は、ライブラリ自体を使用してアクティブなポア数を推定します。このため、フローセルチェックとRun開始時のポア数は、約10％程度の変動が起こります。シークエンシング開始時に報告されたポアの数が大幅に減少している場合は、ライブラリー中の汚染物質がメンブレンを損傷していたり、ポアをブロックしている可能性があります。インプット材料の純度を向上させるために、別のDNA/RNA抽出または精製方法が必要となる場合があります。コンタミネーションの影響は、Contaminants Know-how pieceを参照にして下さい。夾雑物を除去するために別の抽出方法extraction method をお試しください。

MinKNOWのスクリプトに問題

問題点 この問題が生じた可能性のある原因 解決策とコメント
MinKNOW に 「Script failed」と表示されている"
コンピューターを再起動し、MinKNOWを再起動します。問題が解決しない場合は MinKNOW log files MinKNOWログファイルを収集し 、テクニカルサポートにご連絡ください。他のシークエンシングデバイスをお持ちでない場合は、 フローセルとロードしたライブラリーを4℃で保管することをお勧めします。詳細な保管方法については、テクニカルサポートにお問い合わせください。

Pore occupancy below 40%

Observation Possible cause Comments and actions
Pore occupancy <40% Not enough library was loaded on the flow cell Ensure you load the recommended amount of good quality library in the relevant library prep protocol onto your flow cell. Please quantify the library before loading and calculate mols using tools like the Promega Biomath Calculator, choosing "dsDNA: µg to pmol"
Pore occupancy close to 0 The Ligation Sequencing Kit was used, and sequencing adapters did not ligate to the DNA Make sure to use the NEBNext Quick Ligation Module (E6056) and Oxford Nanopore Technologies Ligation Buffer (LNB, provided in the sequencing kit) at the sequencing adapter ligation step, and use the correct amount of each reagent. A Lambda control library can be prepared to test the integrity of the third-party reagents.
Pore occupancy close to 0 The Ligation Sequencing Kit was used, and ethanol was used instead of LFB or SFB at the wash step after sequencing adapter ligation Ethanol can denature the motor protein on the sequencing adapters. Make sure the LFB or SFB buffer was used after ligation of sequencing adapters.
Pore occupancy close to 0 No tether on the flow cell Tethers are adding during flow cell priming (FLT/FCT tube). Make sure FLT/FCT was added to FB/FCF before priming.

予想より短いリード長

問題点 予想される原因 解決策とコメント
予想より短いリード長 DNAサンプルの不要な断片化 読み取り長はサンプルDNA断片の長さを反映します。サンプルDNAは、抽出およびライブラリー調製中の操作で断片化した可能性があります。

1. 抽出の最適な方法については、Extraction Methods の抽出方法を参照してください。

2. ライブラリー調製に進む前に、アガロースゲル電気泳動で、サンプルDNAのフラグメント長の分布を確認してください。 DNA gel2 上の画像では、サンプル1は高分子量ですが、サンプル2は断片化されています。

3. ライブラリー調製中は、試薬を混合するためのピペッティングやボルテックス操作は、プロトコルで指示がないかぎり行わないでください。

利用できないポアの割合が多い場合

問題点 予想される原因 解決策とコメント
利用できないポアの割合が大きい（チャンネルパネルとポアのアクティブポートで青く表示されています）

image2022-3-25 10-43-25 上のアクティブなポアの図は、時間の経過とともに「利用できない」ポアの割合が増加していることを示しています。		 サンプル内に不純物が含まれている 一部のポアに吸着する不純物は、MinKNOWに組み込まれたポアのブロック解除機能によって、ポアから除去することができます。 このステップが完了すると、ポアの状態が「sequencing pore」に戻ります。利用できないポアの部分が多いか、増加した場合:

1.Flow Cell Wash Kit nuclease flush using the Flow Cell Wash Kit (EXP-WSH004) を用いて、ヌクレアーゼ洗浄を 行うことができます。又は
2. PCRを数サイクル実行してサンプルDNAの量を増やし、サンプルDNAに含まれる問題の不純物が相対的に減る（希釈される）ようにします。

Inactiveのポアの割合が高い

問題点 予想される原因 解決策とコメント
利用できない(inactive/unavailable)ポアの割合が高い（チャネルパネルとポアアクティブポートでは水色で表示されています）ポアまたは膜に損傷が起きてしまった。 気泡がフローセルに混入した。 フローセルのプライミングやライブラリーのロードで気泡が入ると、ポアに不可逆的なダメージを与える可能性があります。 推奨の操作方法については、Priming and loading your flow cell のビデオをご覧ください。
利用できないポアの割合が多い場合 サンプルDNAに含まれる不純物 既知の化合物問題で、サンプルDNAに多糖類が含まれた事で、植物のゲノムDNAと結合しポアをブロックした。

1. 植物葉DNA抽出法 Plant leaf DNA extraction methodをご参照ください。
2. QIAGEN PowerClean Pro キットを使用してクリーンアップして下さい。
3. QIAGEN REPLI-g kit.キットを使用して、元のgDNAサンプルで全ゲノム増幅を実行します。
利用できないポアの割合が多い場合 サンプル内に不純物が含まれている 不純物の影響は、 Contaminants の ノウハウを参照して下さい。 サンプルDNAに不純物を残留させないために別の抽出方法をお試しください。

Reduction in sequencing speed and q-score later into the run

Observation Possible cause Comments and actions
Reduction in sequencing speed and q-score later into the run For Kit 9 chemistry (e.g. SQK-LSK109), fast fuel consumption is typically seen when the flow cell is overloaded with library (please see the appropriate protocol for your DNA library to see the recommendation). Add more fuel to the flow cell by following the instructions in the MinKNOW protocol. In future experiments, load lower amounts of library to the flow cell.

温度変動

問題点 予想される原因 解決策とコメント
温度変動 フローセルとデバイスの接続が途切れている。 フローセルの背面にある金属プレートを覆っているヒートパッドがあることを確認してください。 フローセルを再度挿入し、コネクターピンがデバイスにしっかりと接触していることを確認するために軽く押してください。問題が解決しない場合は、テクニカルサービスにご連絡してください。

目標温度に到達しない場合

問題点 予想される原因 解決策とコメント
MinKNOWが "Failed to reach target temperature "(目標温度に達しなかった)と表示する。" 装置が通常の室温より低い場所、または風通しの悪い場所（排気が出来ない場所）に置かれた時にフローセルが過熱してします。 MinKNOWでは、フローセルが目標温度に到達するまでの既定の時間枠があります。時間枠を超えると、エラーメッセージが表示され、シークエンシング実験が続行されます。しかし、不適切な温度でシークエンスを行うと、スループットが低下し、qスコアが低下する可能性があります。シークエンシングデバイスが風通しの良い室温に置かれていることを確認して、MinKNOW再スタートしてください。MinION Mk 1Bの温度制御の詳細については、FAQ を参照してください。

Guppy – no input .fast5 was found or basecalled

Observation Possible cause Comments and actions
No input .fast5 was found or basecalled input_path did not point to the .fast5 file location The --input_path has to be followed by the full file path to the .fast5 files to be basecalled, and the location has to be accessible either locally or remotely through SSH.
No input .fast5 was found or basecalled The .fast5 files were in a subfolder at the input_path location To allow Guppy to look into subfolders, add the --recursive flag to the command

Guppy – no Pass or Fail folders were generated after basecalling

Observation Possible cause Comments and actions
No Pass or Fail folders were generated after basecalling The --qscore_filtering flag was not included in the command The --qscore_filtering flag enables filtering of reads into Pass and Fail folders inside the output folder, based on their strand q-score. When performing live basecalling in MinKNOW, a q-score of 7 (corresponding to a basecall accuracy of ~80%) is used to separate reads into Pass and Fail folders.

Guppy – unusually slow processing on a GPU computer

Observation Possible cause Comments and actions
Unusually slow processing on a GPU computer The --device flag wasn't included in the command The --device flag specifies a GPU device to use for accelerate basecalling. If not included in the command, GPU will not be used. GPUs are counted from zero. An example is --device cuda:0 cuda:1, when 2 GPUs are specified to use by the Guppy command.

Last updated: 11/1/2023

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